A striking feature of Down syndrome (DS; trisomy 21; +21) is the wide range of severity, with strong inter-individual differences in its major features. These include cardiac defects, baseline cognitive function and age-related dementia, as well as several important phenotypes due to altered development and function of blood cells (e.g., childhood leukemias, anemia, autoimmune disorders, and susceptibility to infections). In most cases the genetic or epigenetic factors underlying this variation, and indeed the pathogenesis of the phenotypes themselves, remain largely unknown. Here we hypothesize that the relevant tissues in people with +21 may have accumulated altered patterns of DNA methylation on chromosome 21 and on other chromosomes, potentially affecting some or all of these phenotypes. We have substantial preliminary data supporting this hypothesis, from a profiling method that we developed called MSNP, and from a complementary platform, lllumina Infinium assays. In this highly interactive project we will carry out MSNP and lllumina Infinium assays on peripheral blood leukocyte (PBL) DNAs from people with DS spanning a wide range of ages, including a unique collection of the oldest old survivors, comparing the results with normal controls spanning the same age range. We will validate this epigenetic analysis with bisulfite Pyrosequencing, and correlate the methylation indices and SNP genotypes at the loci with strongest differential methylation with the severity of anemia, autoimmune disorders, and recurrent infections in more than 400 adults with DS. In parallel, we will carry out direct functional studies of the highest priority differentially methylated genes using cell culture and mouse models. While this project is focused on blood cell-related phenotypes, the data may additionally provide a proof-of-principle for future studies of altered DNA methylation in other major organs, including the brain, in this important chromosomal disorder.